Steroid production with streptomyces and cladosporium



I 1 in preparing the novel le-hydroxyprogesterone,we utio r a 1 2,982,694 P Q -l atentedMay 2, 1 9 61 2,982,694 lize as the starting material progesterone which may be STEROID PRODUCTION WITH STREPTOMYCES AND CLADOSPORIUM Thomas H. Stoudt, Westfield, Matthew A. Kozlowski, South River, and William J. McAleer, Roselle, N.J., assignors to Merck & Co., Inc., Rahway, N.J., a corporation of New Jersey No Drawing. Original application Jan. 9, 1958, Ser. No.

' 707,860. Divided and this application Sept. 22, 1958,

Ser. No. 762,273 7 ,3 Claims. ,(Cl. 195-51) The application is a division of our copending application Serial No. 707,860, filed January 9, 1958. This invention is concerned generally with novel steroid compounds and processes of preparing the same. More particularly, it relates to 9a-fluoro-le,l1 3,l7a,21-tetrahydroxy-4-pregnene-3,20-dione, and le-hydroxyprogesterone and to the processes for preparing these compounds.

The novel 9u-fiUOIO-1e,l 118,17a,2l-tetrahydroxy-4-pregnene-3,20-dione and le-hydroxyprogesterone prepared in accordance with this invention possess anti-inflammatory activity and are especially effective for the treatment of arthritis and related diseases.

In preparing the novel 9a-fiuoro-1e,llp,17a,2l-tetrahydroxy-4-pregnene-3,20-dione, we utilize as the starting material the known compound 9a-fiuoro-llfi,l7a,2l-trihydroxy-4-pregnene-3,20-dione having the following structural formula CHzOR In carrying out the process of the present invention,

strains of Streptomyces olivochromogenus, capable ofoxygenation of 9ot-fiuoro-11p,l7a,2l-trihydroxy-4-pregnone-3,20-dione can beobtained from knownvculture collections, for example, one such culture of Streptomyces j olivochromogenus can be'obtained from American Type Culture Collection, Washington, D.C., No. ATCC 13025.

The microorganism is grown under aerobic conditions in intimate contact with 9a fiuoro-11fi,17a,2l-trihydroxy-4 pregnene-3,20-dione ,to-be oxygenated. The fermentation or growing of the microorganismcan be continued until the desired oxygenation has occurred.

represented by the following structural formula enzyme produced by an oxygenating strain of Cladosporipad gesterone, a compound having the structural formulafrom known culture collections; for example, one such culture of Cladosporium sp. can be obtained from Ameri-' can Type Culture Collection, Washington, D .C., No. ATCC 13026. The microorganism is grown under aerobic conditions in intimate contact with the progesterone h to be oxygenated.

The following examples illustrate methods of carrying out the process of the present invention:

EXAMPLE 1 40 Formation of 9a fluoro 1 e,] I 5,] 7u,21 -1etrahydroxy-4- pregnene-3,20-di0ne 80.0 grams of 9a-fiuoro-llfi,l7a,2l-trihydroxy-4 pregnene-3,20-dione 2l-acetate was incubated for 72 hours with Streptomyces olivochromogenus, American Culture Collection No. ATTCC 13025, which had been pregrown for 48 hours in a lactalburnin digest, dextrose, corn steep. liquor medium. The culture filtrate was extracted with ethyl acetate and the; extract. concentrated invacuo at 1 50 45 C; to'a viscous oil. The residue was triturated withpetroleum ether (30-60 C.) to remove excess oils, dissolved in benzene; ethyl acetate (9:1) and charged through a column of diatomaceous earth saturated with water:methanol (1:1). Development of the column with benzenezethyl acetate (9:1) broughtthe 9a-fiuoro-11fl,-

17,2l-trihydroXy-4-pregnene-3,20-dione steroidal substrate off in the first fractions, the 9a-fluoro-1e,1lB,17a,21--

tetrahydroxy-4rpregnene-3,20-dione in the middle frac-' tions mentwith benzene'zethyl acetate (8:2), eluted a' more polar product tentatively identified as 9a-fluoro-6fi,11fi, '1711,2l-tetrahydroXy-4-pregnene-3,20-dione. a

Combination of the middle, fractions yielded-5.9 got crude 1e, 11p],l7a,21-tetrahydroxy-4-pregnene-3,20-dione.

Recrystallization first from acetone and then from meth- V anol yielded 1.9 g. of white crystalline material in the first crop, M.P. 247-250" C., 7 ma 237 my, E, p rcen .425, m 2 (011mm,. (20 carbonyl),,6.02,u (up-unsaturatedketone) um sp. under aerobic conditions to form 1e-hydroxyproand. 9ot-fluoro-l1/3,l7ot,2l-20-tetrahydroxy-4-preg-v 60 nene-3-one iii the final fractions. Subsequent develop- Calculated fOrCg H- O F': C,,63.63 7.32. Found:

Homogeneity was indicated by paper-strip chromatography.

The isolated alcohol 9u-flUO1O-1e,11B,17ot,21-ttrahydroxy-4-pregnene-3,20-dione in pyridine at room temperature for 16 hours'and yielded 9ot-fiUOIO-1e,11fl,17oz, 21 tetrahydroxy-4-pregnene-3,20-dione 1e,21-diacetate. M.P. 218-221 C.,

ALEQ 238 m E, percent, 343

Calculated for C H O F: C, 62.50; H, 6.87. Found: C, 62.63; H, 7.14.

The identity of 9a-fluoro-1e,11B,17u,21-tetrahydroxy-4- pregnene-3,20-dione was established by converting the 90: fluoro 16,1113,l7ec,2l-tetrahydroxy-4-pregnene-3,20- dione le,21-diacetate to 1e-dehydro-9a-fiuoro-11B,17a,21- trihydroxy-4-pregnene-3,20-dione 21-acetate. This was effected by refluxing a portion of the diacetate in glacial acetic acid for 1 hour. Paper-strip chromatographic ex amination of the reaction mixture showed .itto contain principally 1e-dehydro-9a-fiuoro-1 1,8, 170:,2 l-trihydroxy-4- pregnene-3,20-dione 21-acetate contaminated with traces of starting material. The solution was evaporated to dryness in vacuo, the residue chromatographed over acidwashedalumina and the eluted 1e-dehydro-9a-fluoro-1lfl, 17u,21-trihydroxy-4-pregnene-3.20-dione 2l-a'eetate, freed of starting material, was crystallized twice from acetone- Skellysolve B, M.P. 225-236 C.,

X252 238m E, percent, 358

Calculated forC H o Fz' C, 65.64;.H, 6.90. Found: C, 65.92; H, 6.97.

,Mixed melting point with an authentic sample gave no depression, and the infra-red spectra were identical. The Original alcohol 9a-fluoro-le,l113,17a,2l-tetrahydroxy-4- pregnene-3,20-dione can be similarly converted to le-dehydro 9u-fiuoro-l 15,17m,21-trihydroxy-4-pregnene-3,20- dione. tation product is 9u-fluoro-1e,111 17a,21-tetrahydroxy-4- -pregnene-3,20-dione.

i J EXAMPLE 2 V on,

From this evidence it is clear that the fermenpaesaeoai a i for 72 hours. The broth was extracted with 3 x 2 liters of ethyl acetate. The solvent was evaporated, leaving a brown-black oil. The oil was dissolved in 20 ml. of benzene and then partitioned on a 100 gram super-eel column using formamide as the stationary phase and benzene as the mobile phase. The products eluted from the column were unreacted progesterone, 7-hydroxyprogesterone followed by le-hydroxyprogesterone. The benzjenefractions containing the le-hydroxyprogesterone were washed with 100ml. of water to remove formamide. On evaporation of the solvent, le-hydroxyprogesterone was isolated. I 1 l The steroid was characterized by conversion to, the known le-dehydroprogesterone.

It should be understoodthat various changes may be made in the present process as herein described without affecting the results attained. Thus, various modifications of conditions as to time, temperature, alkalinity, acidity, e't'c., and various changes in procedure differing from those herein as illustrative of the preferred embodiments of this invention may be made without departing from the scope thereof. Accordingly, the scope of this invention is'to be determined in accordance with the prior art and the appended claims.

' -We claim:

1. The process for'the production of 1e-hydroxy-4-- pregnene-3,20-dione compounds which comprises subjecting the corresponding 4-pregnene-3,20-dione compound to the activity of an oxidizing enzyme produced by an oxygenating strain of a microorganism selected from the group consisting of Streptomyces olivochromogenus and Cladosporium sp. under aerobic conditions.

2. The process for the production *of 9u-fluoro-1e,11p, 1711,21 tetrahydroxy-4-pregnene-3,20-dione which comprises subjecting 9 t-fiuoro-l1,6,l7a,21-trihydroxy-4-pregnene3,20-dione to the action of an oxidizing enzyme produced by an oxygenating strain of Streptomyces olivochromogenus under aerobic conditions. 7

3. The process for the production of le-hydroxyprogesterone which comprises subjecting progesterone to the action of an oxidizing enzyme produced by an oxygenating strain of Cladosporium sp. under aerobic conditions.

References Cited in the file of this patent UNITED STATES PATENTS OTHER. REFERENCES;

Ann. New York Acad. Sciences, vol. 60,1, pp.

27 and 150. 

1. THE PROCESS FOR THE PRODUCTION OF 1E-HYDROXY-4PREGNENE-3,20-DIONE COMPOUNDS WHICH COMPRISES SUBJECTING THE CORRESPONDING 4-PREGNENE-3,20-DIONE COMPOUND TO THE ACTIVITY OF AN OXIDIZING ENZYME PRODUCED BY AN OXYGENATING STRAIN OF A MICROORGANISM SELECTED FROM THE GROUP CONSISTING OF STREPTOMYCES OLIVOCHROMOGENUS AND CLADOSPORIUM SP. UNDER AEROBIC CONDITIONS. 